Zhang, Y. and C.A. Suttle. 1994. Design and use of PCR primers for B-family DNA polymerase genes to detect and identify viruses and microbes. Abstracts American Society Limnology and Oceanography, and Phycological Society American, Miami, FL, June 1994.

B-family DNA polymerases are found in eukaryotes, prokaryotes and dsDNA viruses. The polymerase gene includes motifs coding for both highly conserved and variable domains of amino acid sequences. Moreover, DNA polymerase sequences are highly conserved at the amino acid level but not at the nucleotide level; consequently, the polymerase gene is an ideal target for which PCR primers of wide-ranging specificity can be designed. The DNA-polymerase gene of a large dsDNA virus that infects the photosynthetic picoflagellate Micromonas pusilla was sequenced. Our goals were to compare the inferred amino acid sequence with those for viruses which infect a Chlorella-like alga that is found as an endosymbiont in Paramecium bursaria, in order to determine if the viruses are related, and if so, to identify regions of the gene that could be used for designing algal-virus-specific PCR primers. We identified strings of 11 and 9 amino acids that were completely conserved among Micromonas and Chlorella viruses but which were not found in any other sequences in GenBank, implying that the viruses are evolutionarily related. PCR primers designed from these data have been used to amplify DNA polymerase gene sequences from 3 groups of viruses infecting Chlorella sp., Micromonas pusilla and Chrysochromulina brevifilum. The DNA polymerase gene is an ideal target for amplification with PCR primers. By varying degeneracy it is possible to design primers that can be used to detect, identify and ultimately quantify a wide range of microbes and viruses.